Progress during FY18: 1) Progress in GESTALT - Sample collection of 11 blood cell types continues. - Completed monocyte ATAC-seq sample collection, library preparation and sequencing. Data analysis is ongoing. - B cell ATAC-seq sample collection completed. Library preparation and sequencing is ongoing. - Initiated cell-activation studies in T cells and monocytes. We tested cell stimulation reagents and compared responses in total PBMC to purified CD4+ T cells and monocytes. We developed a cytokine panel to probe many cytokines simultaneously by multi-parameter flow cytometry. Development of a phospho-flow panel is almost complete. - Initiated single-cell RNA-Seq studies. - Initiated analysis of tissue-specific DNA methylation using first two sets of GESTALT donors. 2) Progress in mitochondrial and autophagy studies in CD4+ T cells. Our aim was to detect mitophagy in human primary CD4+ T cells and search for any measurable differences between young and old using flow cytometry. A mitophagy detection kit was used for this. This kit is composed of Mtphagy Dye, a reagent for detection of mitophagy, and Lyso Dye. Mtphagy Dye has fluorescent properties for detecting mitochondrial acidification during mitophagy in the long-wavelength region that does not damage mitochondria. We performed a total of six different experiments. For the first five experiments we followed the procedures of the manufacturers manual. For four of these experiments, we used previously isolated and cryo-frozen CD4+ cells, and for one of the experiments we used freshly isolated CD4+ cells. During these experiments, CCCP reagent was used as an inducer, while Bafilomycin was used as an inhibitor. The kit is specifically designed for HeLa cells and to show results with immunofluorescent microscopy- confocal. Our results were not as satisfactory as desired. Therefore, for the sixth experiment, we modified the experimental protocol slightly in two sets and compared the results to two sets without modification to eliminate technical or experimental differences. For the comparison, we used a total of four sets (two sets modified and two sets original protocol) prepared from cryo- frozen CD4+ cells from a young donor and four sets (again two sets modified and two sets original protocol) prepared from old donor cryo- frozen CD4+ cells. With the modified protocol, we saw noticeable differences between young and old. We need to run more samples from young and old donors frozen or/and freshly isolated CD4+T cells. For this study, we used seven cryo-frozen samples and each sample had about 7 million CD4+T cells, while one sample was of freshly isolated CD4+T cells. 3) Analysis of autoantibody production in humans (collaboration with Erika Darrah and Antony Rosen). We cloned IgH and IgL chains from single cell plasmablasts from rheumatoid arthritis (RA) patients. Sera from these patients was bound to PAD4-coupled matrices and adsorbed antibodies evaluated by mass spectrometry in collaboration with Dr. Gregory Ippolito (UT Austin). Comparison of mass spec and DNA sequence data revealed a unique CDR3 determinant that was present in patient sera. Ongoing studies are aimed at understanding the significance of this epitope in RA.